1

SIRT3 regulates α-SMA production through the succinate dehydrogenase-

GPR91 pathway in hepatic stellate cells

Ying Hui Li‡, Dae Hee Choi

‡, Eun Hye Lee

§, Su Ryeon Seo

§, Seungkoo Lee

¶, Eun-Hee Cho

‡1

‡Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon,

200-701, Korea §Department of Molecular Bioscience, College of Biomedical Science, and Institute of Bioscience&

Biotechnology, Kangwon National University, Chuncheon, 200-701, Korea ¶Department of Anatomic Pathology, School of Medicine, Kangwon National University, Chuncheon,

200-701, Korea

*Running Title : SIRT3 regulates hepatic stellate cell activation

1Corresponding author: Eun-Hee Cho

Department of Internal Medicine, School of Medicine, Kangwon National University, 26 Kangwondaehak-gil, Chuncheon-si, Gangwon-do, 200-701, Korea Tel: +82-33-258-9167 Fax: +82-33-258-2455 E-mail:ehcho@kangwon.ac.kr

Keywords : Sirtuin 3, succinate dehydrogenase, GPR91, hepatic stellate cell, nonalcoholic fatty liver disease, honokiol

ABSTRACT Sirtuin 3 (SIRT3) is an NAD

+-dependent

protein deacetylase. Recent studies have shown that SIRT3 expression is decreased in nonalcoholic fatty liver disease (NAFLD). Moreover, SIRT3 is a key regulator of succinate dehydrogenase (SDH), which catalyzes the oxidation of succinate to fumarate. Increased succinate concentrations and the specific G protein-coupled receptor 91 (GPR91) are involved in the activation of hepatic stellate cells (HSCs). In this study, we aimed to establish whether SIRT3 regulated the SDH activity, succinate, and GPR91 expression in HSCs and an animal model of NAFLD. Our goal was also to determine whether succinate released from hepatocytes regulated HSC activation. Inhibiting SIRT3 using SIRT3 siRNA exacerbated HSC activation via the SDH-succinate-GPR91 pathway and SIRT3 overexpression or honokiol treatment attenuated HSC activation in vitro. In isolated liver and HSCs from methionine- and choline-deficient (MCD) diet-induced NAFLD, the expression of SIRT3 and SDH activity was decreased and the succinate concentrations and GPR91 expression were increased. Moreover, we found that GPR91 knockdown or resveratrol treatment

improved the steatosis in MCD diet-fed mice. The present investigation revealed a novel mechanism of SIRT3-SDH-GPR91 cascade in MCD diet induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies aimed at targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD treatment. INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in many developed countries (1) and nonalcoholic steatohepatitis (NASH), the more severe histological form of NAFLD, is associated with an increased risk for the progression to cirrhosis in 20% of these patients (2). NAFLD also increases cardio-metabolic risk (3-5) and all-cause mortality (6,7) in humans. It is presently regarded as the main cause of cryptogenic liver cirrhosis in the United States (8). During liver injury, quiescent hepatic stellate cells (HSCs) transdifferentiate into activated myofibroblasts which produce alpha-smooth muscle actin (α-SMA) and become a major cell type in hepatic fibrogenesis (9,10).

Sirtuin 3 (SIRT3) is an NAD+-

dependent protein deacetylase, predominantly

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.692244The latest version is at JBC Papers in Press. Published on February 24, 2016 as Manuscript M115.692244

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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localized in the mitochondrial matrix (11-13). SIRT3 is upregulated during the prolonged fasting or a calorie restricted diet and is thus involved in the metabolic regulation of obesity and diabetes (14-16). Based on several recent studies, SIRT3 is a primary regulator of the acetylation of mitochondrial proteins and their biological activity (16-19) and is associated with NAFLD (20-22).

Two studies yielded findings showing that that SIRT 3 is a major physiological regulator of succinate dehydrogenase (SDH) activity (23,24). SDH catalyzes the oxidation of succinate to fumarate thereby decreasing SDH activity resulting in increased succinate levels (25,26). The succinate receptor (also known as GPR91) is a G protein-coupled receptor expressed in various tissues, including retina, liver, and kidneys (27-31). Locally increased succinate levels and GPR91 activation have recently emerged as novel signaling molecules in local stress situations (25).

In a previous study, we showed that decreased SDH activity led to increased cellular succinate levels and succinate receptor (GPR91) overexpression with increased α-SMA production in the isolated HSCs of MCD diet-induced NASH mice (32). These observations led us to question whether SIRT3 expression could modulate HSC activation through SIRT3-SDH-GPR91 signaling in NASH. To the best of our knowledge, the role of SIRT3 in the regulation of HSC activation has not been fully characterized. In this study, we evaluated the effects of SIRT3 on GPR91 regulation through SDH to mitigate the progression of NASH in HSCs and animal model and we determined whether succinate secreted from hepatocytes regulated HSC activation.

EXPRERIMENTAL PROCEDURES Materials - Overexpression of α-SMA, a

hallmark of myofibroblastic transdifferentiation, was used as a marker for HSC activation (33,34). Dulbecco’s modified Eagle’s media (DMEM), completely deficient of methionine and choline (MCD media) and methionine- and choline supplement (MCS media, control media) were purchased from WELGENE (Kyeongsan, Korea). Palmitate was purchased from Sigma (St. Louis, MO, USA). The AAV- GPR91 shRNA (Vector Biolabs, Philadelphia, PA, USA) or the AAV6-GFP shRNA (Vector Biolabs,

Philadelphia, PA, USA) were used for viral production.

Cell Culture - LX2 cells are immortalized human stellate cells and were provided by Prof. Ja June Jang, Seoul National University. The cells were cultured in DMEM with 10% fetal bovine serum (FBS) supplemented with 1% penicillin/streptomycin antibiotic solution. AML12 cells were cultured in DMEM F12 media (WELGENE, Kyeongsan, Korea) supplemented with 10% FBS, and 1% penicillin/streptomycin antibiotic solution. Cells were maintained in a humidified 37

o C incubator

with 5% CO2. Western Blot Analysis - Whole cells were

lysed in radioimmunoprecipitation (RIPA) buffer containing 25 mM Tris-HCL (pH-7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1% SDS, and protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany) on ice. Equal amounts of proteins were resolved on a SDS/PAGE and then electro-transferred onto PVDF membranes and blocked with 5% nonfat dry milk for 30 min at room temperature. Levels of proteins were determined by incubation with primary antibodies at appropriate dilutions. Primary antibodies included those specific to GPR91 (sc-50466, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ERK1/ERK2 (MAB1576, R&D Systems, Minneapolis, MN, USA), phospho-ERK1/ERK2 (AF1018, R&D Systems), SIRT3 (#2627, Cell Signaling, Danvers, MA, USA), α-SMA (GTX112861, GeneTex, Irvine, USA), and GAPDH (GTX627408, GeneTex). The bound antibodies were further incubated with secondary antibodies conjugated to HRP, detected with the Westsave Star Detection Reagent system (AbFrontier, Seoul, Korea).

SIRT3 siRNA Transfection and Adenoviral Transfection of SIRT3 - LX2 cells grown in the exponential phase were seeded in a 6-well plate and were then transfected with 100 nM SIRT3-targeted siRNA (sc-61555, Santa Cruz, Biotechnology) or nontargeting RNA for 6 h using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Adenovirus transfection was performed when human LX2 cells reached 30-40% confluency in a 6-well plate. Before transfection, LX2 cells were changed using either control or MCD media and then infected with human SIRT3 adenovirus

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(Ad-SIRT3, #1499, Vector Biolabs, Malvern, PA, USA) or control adenovirus expressing

LacZ (Ad-CMV- 𝛽-gal, #1080, Vector Biolabs) at a MOI of 30. At 24 h post-infection, cells were lysed and subjected to western blotting. Some LX2 cells were infected with human Ad-SIRT3 or control adenovirus expressing LacZ (Ad-LacZ) at a MOI of 30. At 8 h post-transfection, cells were stimulated with palmitate for 20 h, after which total proteins were extracted.

Description of Animals and Isolation of HSCs and Hepatocytes - Male C57BJ6 mice, 6-to 8-week-old and weighing 18–20 g were purchased from Central Animal Laboratory (Korea). All mice were housed at ambient temperature (22 ± 1°C) with a 12/12-h light/dark cycle and free access to water and food. The mice were fed the methionine- and choline- deficient diet (MCD diet group) as an animal model of NAFLD, or control chow diet (control group) for 4 weeks. All mice were fed their assigned MCD diet for 4 weeks and the AAV6-GFP shRNA (4x10

11 PFU, n = 4) or AAV-

GPR91 shRNA (4x1011

PFU, n = 8) were injected via the tail vein on the first day of MCD diet feeding. In another group, mice received 100 mg/kg/day of resveratrol daily with the MCD diet for 4 weeks (n = 8) and C57BL/6 mice on a regular chow diet were used as the control group (WT control, n = 4). Primary mouse HSCs and hepatocytes were isolated from the livers of mice (10 to 12 weeks old) by in situ pronase E and collagenase B perfusion followed by density gradient centrifugation. Primary cells were >95% pure. Cells were grown in standard tissue culture plastic dishes in DMEM media with 10% FBS and antibiotics. Primary cells were incubated at 37°C and used 3 days after plating.

Succinate Dehydrogenase Assay and Succinate Assay - The SDH assay was performed using the ab109908-Complex II enzyme activity microplate assay kit (Biovision, Milpitas, CA, USA). Cell lysate (5 μL) was added to a mixture containing SDH assay buffer, SDH substrate mix, and SDH probe. Absorbance readings at 600 nm were taken every 20 s for a total of 60 min. The data are expressed as mOD.min

−1. The level of cellular

succinate was determined with a succinate colorimetric assay kit (BioVision). Succinate levels were read at 450 nm with each

measurement performed in triplicate. Deacetylation Assay - SDH subunit A

(SDHA) was immunoprecipitated from the total cell lysate with SDHA (sc-166909, Santa Cruz Biotechnology) antibody. Then, western blots were probed with anti-acetylated lysine antibody (#9441S, Cell Signaling).

RT-PCR - Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). Primers were designed as follows: SIRT3 (5-CGT CAC TCA CTA CTT TCT CC-3) and (5-ACC ACA TGC AGC AAG AAC CT-3), GPR91 (5-GCA TGT GTC TAA CAC TGT TG-3) and (5-CTT CTG TCC CAA CTA CTG TG-3), α-SMA (5-CCA CCG CAA ATG CTT CTA AGT-3) and (5- GGC AGG AAT GAT TTG GAA AGG-3), TGF-β1 (5-TCG ACA TGG AGC TGG TGA AA-3) and (5-GAG CCT TAG TTT GGA AGA TCT G-3), collagen a1 (5-GAA CGC GTG TCA TCC CTT GT-3) and (5-GAA CGA GGT AGT CTT TCA GCA ACA-3), and GAPDH (5-GGC ATG GAC TGT GGT CAT GAG-3) and (5-TGC ACC ACC AAC TGC TTA GC-3). cDNA was synthesized by reverse transcription with the PrimeScript 1

st strand

cDNA Synthesis Kit (Takara Bio, Shiga, Japan) and amplified by PCR with Maxima SYBR Green/ROX qPCR Master Mix (2x) (Thermo) using standard protocols. All amplified products were assessed with 2% agarose gel electrophoresis and photographed using ultraviolet illumination.

Hepatic TG Measurement - TG contents in the liver were determined using the Triglyceride Reagent kit (T2449, Sigma, St. Louis, MO) as described by the manufacturer.

Histological Analysis and Immunohisto-Chemistry - Samples of mouse liver were fixed in 10% (w/v) phosphate-buffered formalin for 18–20 h. After dehydration through a graded series of ethanol solutions, the tissues were embedded in paraffin wax. Serial frontal sections were cut at 5 μm intervals and stained with hematoxylin and eosin (H&E), and Masson's trichrome. The obtained 5-μm sections were deparaffinized in xylene and rehydrated through a graded ethanol series into water. Endogenous peroxidase activity was blocked in 3% H2O2. The slides were subsequently placed on a Dako Autostainer immunostaining system for use in immunohistochemistry analyses using polyclonal antibodies against GPR91(1:100,sc-50466, Santa Cruz Biotechnology), SIRT3

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(1:200, #2627, Cell Signaling) α-SMA(1:500, GTX112861, GeneTex) and incubated for 5 h. In the next step, the slides were blocked for endogenous biotin with an avidin-biotin blocking system (Dako, X0590). Labeled streptavidin-biotin complex plus (Dako, K0675) served as the detection system and hematoxylin was used for counterstaining.

Immunofluorescence Microscopy - To locate the mitochondria, heat shock protein 60 (HSP 60) was used to co-label the samples. For double staining of SIRT3 and HSP 60, sections were stained with primary antibodies (both 1:200, sc-49744, sc-13966 Santa Cruz Biotechnology), and then with goat anti-rabbit FITC-conjugated secondary antibody (1:200 #31635 Invitrogen) and donkey anti-goat Cy3- conjugated secondary antibody (1:200 ab6949, Abcam, Cambridge MA USA) for 1 h. Hoechst 33342 staining for 10 mins was used to evaluate chromosomes. After three washes, liver samples were mounted in anti-fade medium (Vectashield, Burlingame, CA, USA), and examined under a laser scanning confocal microscope (LSM 710, Zeiss, Germany).

Statistical Analyses - All data are expressed as means ± SEM from at least three independent experiments and GAPDH was used as a loading control. Data analyses for the two groups were performed using the t-test with p- values < 0.05 indicating statistical significance.

RESULTS Succinate, SDH Inhibitor and Fumarase

Inhibitor Activates HSCs - To investigate the role of succinate in HSC activation, we treated HSCs with succinate and demonstrated that succinate itself increased the expression of GPR91, ERK phosphorylation, and α-SMA production in HSCs (Fig. 1A). However, pretreatment with 10 µM U0126 (ERK inhibitor) significantly blocked the succinate-induced upregulation of GPR91 and α-SMA expression in the LX2 cells. These findings suggest that the ERK pathway is downstream of the succinate pathway. Treatment with malonate, a known SDH inhibitor, showed increased expression of GPR91 and α-SMA production in HSCs (Fig. 1 B) and decreased SDH activity and increased succinate concentrations (Fig. 1C, D). Treatment with D-malate, a fumarase inhibitor, increased GPR91 and α-SMA production in HSCs (Fig. 1E), decreased fumarase and SDH activity

(Fig 1F, G), and increased succinate concentrations (Fig 1H).

SIRT3 siRNA Transfection Activates HSCs -To investigate the role of SIRT3 in HSC activation, we used siRNA to deplete SIRT3 in HSCs. SIRT3 siRNA transfection increased the mRNA levels of GPR91, α-SMA, TGF β1, and collagen type 1 (Fig. 2A, B). SIRT3 siRNA transfection increased the expression of the mRNA levels of GPR91 suggesting a negative association of SIRT3 and GPR91 in HSCs activation.

SIRT3 Regulates SDH Activity and GPR91 Expression in HSCs - LX2 cells treated with SIRT3 siRNA for 24 h demonstrated decreased expression of SIRT3 and increased protein expression of GPR91 and α-SMA compared with control siRNA treatment (Fig. 3A). Moreover, LX2 cells treated with SIRT3 siRNA for 24 h demonstrated decreased SDH activity and increased succinate concentrations (Fig. 3B, C). SIRT3 siRNA treatment in LX2 cells induced decreased SIRT3 expression and increased acetylation of SDHA in immunoprecipitations (Fig. 3D). LX2 cells treated with adenoviral transfection of SIRT3 overexpression for 24 h showed increased expression of SIRT3 and decreased expression of GPR91 and α-SMA compared with a control adenoviral transfection (Fig. 3E). Additionally, LX2 cells transfected with adenoviral SIRT3 overexpression showed increased SDH activity and decreased succinate concentrations in HSC lysates (Fig. 3F, G). SIRT3 overexpression in LX 2 cells increased SIRT3 production and decreased acetylation of SDH subunit A (SDHA) in immunoprecipitation (Fig. 3H). These results suggest that SIRT3 regulates SDH activity and cellular succinate levels leading to HSC deactivation, through inhibition of GPR91. Both SIRT3 silencing and overexpression modulate MAP kinase as evidenced by SIRT3 siRNA-induced increase in phosphorylation of ERK (Fig. 3A) or the Ad-SIRT3-induced decrease in ERK phosphorylation (Fig. 3E).

Effects of Palmitate, MCD Media on SIRT3, Succinate and GPR91 in HSCs - We tested whether SIRT3 expression was affected by palmitate or MCD media treatment in HSCs. When LX2 cells were incubated with palmitate for 20 h, this led to decreased protein expression of SIRT3 and increased expression of GPR91 and α-SMA (Fig. 4A), as well as decreased

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mRNA expression of SIRT3 in cell lysates compared with control treatment (Fig. 4B). We used MCD media instead of the MCD diet which is a widely used technique to create an animal model of nonalcoholic steatohepatitis (NASH), to investigate the influence of MCD media on the SIRT3, SDH and GPR91 pathway in HSCs. We cultured LX2 cells in MCD media for 24 h and monitored SIRT3, GPR91, and α-SMA expression in the LX2 cells after treatment. LX2 cells incubated with MCD media for 24 h showed decreased protein expression of SIRT3, and increased expression of GPR91 and α-SMA (Fig. 4C). Additionally, LX2 cells incubated with MCD media for 24 h demonstrated decreased mRNA expression of SIRT3 in HSC lysates compared with control treatment (Fig. 4D). These results indicate that palmitate and MCD media can induce activation of HSCs through SIRT3 deactivation and GPR91 activation.

SIRT3 Overexpression Attenuates Palmitate- and MCD Media-induced HSC Activation - To test whether SIRT3 could improve palmitate or MCD media- induced HSC activation, LX2 cells were infected with Ad-SIRT3 or Ad-control and subsequently treated with palmitate (300 μM) for 20 h. Palmitate treatment significantly decreased SIRT3 expression (Fig. 5A) and increased GPR91 and α-SMA protein expression in the LX2 cells infected with Ad-control (Fig. 5A). However, GPR91 and α-SMA protein expression was attenuated in the LX2 cells infected with Ad-SIRT3 in the presence of palmitate (Fig. 5A). We also found that SIRT3 adenoviral overexpression ameliorated the palmitate-induced decrease in SDH activity and the palmitate-induced increase of succinate concentrations (Fig. 5B, C). MCD media treatment significantly increased GPR91 and α-SMA protein expression in the LX2 cells infected with Ad-control (Fig. 5D). However, GPR91 and α-SMA protein expression were decreased in the LX2 cells infected with Ad-SIRT3 in the presence of MCD media (Fig. 5D). Overexpression of SIRT3 ameliorated reduction of SDH activity and attenuated increased concentrations of succinate by MCD media (Fig. 5E, F). We further found that when LX2 cells were incubated with Ad-SIRT3, phosphorylation of ERK was attenuated in the presence of palmitate or MCD media (Fig. 5A, D).

Honokiol treatment Attenuates Palmitate-

and MCD Media-induced HSC Activation - To test whether honokiol, a natural biphenolic compound, could improve palmitate- or MCD media- induced HSC activation, LX2 cells were treated with or without honokiol (10 μM). After 4 h, the cells were incubated with or without palmitate (300 μM) for 20 h. Palmitate treatment significantly decreased SIRT3 expression (Fig. 6A) and increased GPR91 and α-SMA protein expression in the LX2 cells treated with control (Fig. 6A). However, GPR91 and α-SMA protein expression was attenuated in the LX2 cells treated with honokiol in the presence of palmitate (Fig. 6A). We also found that honokiol treatment increased SIRT3 expression (Fig. 6A) and ameliorated the palmitate-induced decrease in SDH activity and the palmitate-induced increase of succinate concentrations (Fig. 6B, C). MCD media treatment significantly increased GPR91 and α-SMA protein expression in the LX2 cells (Fig. 6D). However, GPR91 and α-SMA protein expression were decreased in the LX2 cells treated with honokiol in the presence of MCD media (Fig. 6D). Honokiol treatment attenuated the MCD media-induced decrease in SIRT3 expression (Fig. 6D) and ameliorated reduction of SDH activity and attenuated increased concentrations of succinate by MCD media (Fig. 6E, F). We further found that when LX2 cells were incubated with honokiol, phosphorylation of ERK was attenuated in the presence of palmitate or MCD media (Fig. 6A, D).

AAV-GPR91 sh RNA Knockdown and SIRT3, GPR91 and α-SMA Expression in Isolated HSCs and Liver from MCD diet-fed Mice as a Model of NAFLD - In a previous study, we demonstrated overexpression of GPR91 protein, decreased SDH activity and increased succinate concentrations in isolated HSCs of MCD diet-fed mice as a model of NAFLD (32). In this study, we applied a novel gene therapeutic approach using recombinant adeno-associated virus (AAV)-mediated RNA knockdown of GPR91 gene expression in a mouse model of NAFLD.

All mice were fed their assigned MCD diet for 4 weeks and the AAV6-GFP shRNA (control shRNA) or AAV-GPR91 shRNA were injected in C57BL/6 mice via the tail vein on the first day of MCD diet feeding. In MCD diet-fed mice treated with AAV-GPR91 shRNA, we noted a decrease in steatohepatosis in H&E staining (Fig.

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7A) and decreased fibrosis in Masson’s trichrome staining (Fig. 7B) compared with the AAV-GFP shRNA treatment. Immunostaining for GPR91, and α-SMA confirmed the western blot results, which revealed that AAV-GPR91 shRNA knockdown decreased the amount of GPR91 and α-SMA positive cells (Fig. 7C, D).

In the liver and isolated HSCs from mice that were fed the MCD diet, which was injected with control shRNA (AAV6-GFP shRNA), the expression of SIRT3 was not changed and the expression of GPR91 and α-SMA protein were increased compared with those from mice that were fed the MCD diet, which was injected with AAV6-GPR91 shRNA. On the other hand, the data pertaining to the AAV-GPR91 shRNA treated mice showed decreased GPR91 and α-SMA protein expression (Fig. 7E, F). To explore a possible mechanism by which GPR91 ameliorated hepatic steatosis, we conducted a western blot analysis, which revealed that GPR91 knockdown with AAV-GPR91 shRNA treatment decreased ERK phosphorylation in the liver and isolated HSCs from the MCD diet-fed mice (Fig. 7E, F).

Based on these results, we suggest that GPR91 knockdown improved steatohepatitis and decreased HSC activation through decreased ERK phosphorylation.

Resveratrol Treatment and SIRT3, GPR91 and α-SMA Expression in Isolated HSCs and Liver from MCD diet-fed Mice as a Model of NAFLD - To examine its physiological relevance in vivo, we tested whether SIRT3 expression with resveratrol was attenuated in the liver and isolated HSCs of MCD diet-fed mice as a model of NAFLD.

Resveratrol treatment prevented hepatic steatosis and fibrosis induced by a MCD diet (Fig. 8A, B). Immunostaining for GPR91 and α-SMA showed that the MCD diet increased the amount of GPR91 and α-SMA positive cells and resveratrol treatment in mice that fed the MCD diet decreased the amount of GPR91 and α-SMA positive cells (Fig. 8C, D). Resveratrol treatment increased SRT3 protein expression and decreased GPR91 and α-SMA protein expression in the liver and isolated HSCs of MCD diet-fed mice (Fig. 8E, F). Immunostaining for SIRT3 showed that the MCD diet decreased the number of SIRT3 positive cells (Fig. 9A) and resveratrol treatment of MCD diet-fed mice showed increased

number of SIRT3 positive cells (Fig. 9A). To further determine the subcellular localization of SIRT3 in cells, we co-stained with a mitochondria-specific HSP 60 (green) with SIRT3 (red) and the merged image showed complete overlapping of the two signals (Fig. 9B), indicating that SIRT3 exclusively localized to mitochondria.

Hepatic triglycerides were increased in mice fed the MCD diet compared with mice fed the chow diet but were decreased significantly in MCD diet-fed mice with AAV-GPR91 shRNA knockdown and MCD diet-fed mice with resveratrol treatment (Fig. 9C).

Thus, resveratrol treatment decreased the accumulation of lipid droplets by increasing SIRT3 activity and decreasing GPR91 and α-SMA protein levels.

The Effect of Palmitate and MCD Media Treatment on Hepatocytes Isolated from Chow diet-fed Mice - Palmitate treatment on isolated hepatocytes from chow diet-fed mice decreased the SIRT3 expression in primary hepatocytes (Fig. 10A). Palmitate treatment of isolated hepatocytes from chow-diet-fed mice demonstrated decreased the SDH activity and increased succinate concentrations in cell lysates (Fig. 10B, C). Primary hepatocytes from chow diet-fed mice exposed to MCD media also showed decreased SIRT3 protein expression (Fig. 10D). Additionally, MCD media treatment of primary hepatocytes showed decreased SDH activity and increased succinate concentrations in cell lysates (Fig. 10E, F). These findings indicate that when primary hepatocytes are subjected to palmitate or MCD media, this results in decreased SDH activity, increasing the cellular succinate concentrations.

The Effect of Conditioned Media from Hepatocytes Exposed to Palmitate or MCD Media on HSC Activation In Vitro - We previously demonstrated that palmitate or MCD media directly activated HSCs via decreased SDH activity, thus increasing succinate levels and inducing GPR91 activation (32). To establish whether the activation of HSC was induced indirectly through the paracrine action of hepatocytes, we treated mouse hepatocytes (AML12 cells) with palmitate (300 μM) for 20 h. The conditioned media (CM) from palmitate-treated hepatocytes was transferred to LX2 cells (Fig. 11A). We first measured SDH activity and succinate concentrations of the CM from

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palmitate-treated hepatocytes and demonstrated that SDH activity decreased significantly while succinate concentrations increased in the CM from palmitate-treated hepatocytes, when compared with control-treated hepatocytes (Fig. 11B, C). In addition, the CM from palmitate-treated hepatocytes decreased the protein expression of SIRT3 and increased expression of GPR91 and α-SMA proteins in LX2 cells (Fig. 11D). The CM from palmitate-treated hepatocytes was shown to decrease the SDH activity and increase the succinate concentrations in whole cell lysates of LX2 cells (Fig. 11E, F).

DISCUSSION

In this study, we provided several lines of evidence that implicate SIRT3 in SDH activity and GPR91 expression in HSC activation in NAFLD. First, succinate and SDH activity activate HSCs. Second, inhibiting SIRT3 with SIRT3 siRNA induced decreased SDH activity, increased succinate concentrations in whole cell lysates, and increased protein expression of GPR 91 and α-SMA in LX2 cells, demonstrating HSCs activation through the SIRT3-SDH-GPR91 pathway. Third, SIRT3 overexpression with adenoviral SIRT3 transfection or honokiol treatment ameliorated palmitate-induced and MCD media-induced HSC activation through the SIRT3-SDH-GPR91 pathway. Fourth, we demonstrated that GPR91 knockdown with AAV-GPR91 shRNA or nonspecific SIRT3 activation with resveratrol improved steatosis and decreased α-SMA production in MCD diet-fed mice. Fifth, increased concentrations of succinate in the cell lysates of primary hepatocytes exposed to palmitate or MCD media and CM of palmitate-treated hepatocytes may indirectly enhance the activation of HSCs suggesting that succinate acts as a paracrine modulator between hepatocytes and HSCs.

To the best of our knowledge, this is the first reported study to examine the association of SIRT3 and HSC at the cellular level. SIRT3 is the primary mitochondrial enriched deacetylase (35).

SIRT3 KO high fat diet-fed mice were reported to exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake (36). Chronic high fat feeding for 12 weeks was found to result in decreased SIRT3 mRNA levels and SIRT3 protein expression compared

with chow-fed controls (37). Our study demonstrated for the first time that SIRT3 siRNA transfection in HSCs induced decreased SDH activity thereby increasing succinate-GPR91 signaling, ultimately resulting in the activation of HSCs.

In a previous study, we demonstrated that the succinate receptor, GPR91 is overexpressed in HSC activation and GPR91 siRNA treatment in LX2 cells showed decreased α-SMA protein expression (32). In this study, we demonstrated that GPR91 knockdown by GPR91 shRNA tail vein injection decreased α-SMA expression in the liver, isolated HSCs and ameliorated the NASH phenotype in MCD diet-fed mice suggesting that GPR91 could be a therapeutic target for the treatment of NAFLD.

Constant with previous studies, we demonstrated that SDH, complex II of the electron transport chain, is regulated by SIRT3 (23,24). Thus, while SIRT3 increases SDH activity and decreases succinate concentrations, these findings differ from those reported in other studies (35,37). The conflicting results regarding the relationship of SDH and SIRT3 are currently not well understood and this warrants further investigation. Nonetheless, the data reported here demonstrated that SIRT3 regulated SDH activity, thereby decreasing cellular succinate concentrations. Moreover, decreased succinate concentrations deactivate GPR91 expression, resulting in HSC deactivation, suggesting SIRT3 as a drug target for a HSC deactivator.

Succinate is an important metabolic intermediate that constitutes one of the intermediates of the citric acid cycle and is regarded as a signaling molecule (25,27,38), acting by binding to the succinate receptor, GPR91. There have been several reports showing that succinate and GPR91 are involved in renin release (39-42) in the kidney. However, few studies have explored any association between succinate metabolism and metabolic syndromes including NAFLD. Correa et al. demonstrated that succinate levels in the perfusate effluent of rat liver were rapidly increased up to 14-fold after ischemia was induced by interrupting portal flow. They also reported that succinate accelerated HSC activation (43). The current study confirmed that succinate and GPR91 activation induced HSC activation and GPR91 knockdown, using AAV-GRP91 shRNA, ameliorated HSC activation and

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the NASH phenotype of MCD diet-fed mice. A Pharmaceutical or nutriceutical activator

of SIRT3 is an area of great interest. However, to date, no known SIRT3-specific agonist or antagonist has been developed. Resveratrol is a natural polyphenol, found mainly in grape skin and berries, which serves as a calorie restriction mimetic. In 2003, Howitz et al. reported for the first time that resveratrol, which is also a polyphenol in red wine, was a small molecule activator of sirtuin 1 (SIRT1) (44). Resveratrol regulates AMPK and SIRT1 directly and indirectly in concert to promote protection against metabolic disease, mimicking calorie restriction; resveratrol also inhibits phosphodiesterases (PDEs) and the nuclear factor-kappaB (NF-κB) family of transcription factors (45). Some papers have shown that resveratrol did not activate SIRT1 in vitro in the presence of p53 and PGC1α, calling into question its ability to directly activate SIRT1 (46, 47). Based on these findings, the possibility has been raised that resveratrol could have health benefits through other unknown mechanism(s) apart from SIRT1 activation.

Resveratrol prevented high-fat diet induced hepatic steatosis and endoplasmic reticulum stress in rats (48). In a randomized double-blind controlled study, resveratrol for 30 days in obese humans demonstrated decreased intrahepatic lipid contents and increased intramyocellular lipid levels. This resulted in activated AMPK, increased SIRT1, and PGC-1α protein expression, as determined by musc le biopsies (49). In another study, it was showed that resveratrol acted as a direct stimulator of complex 1 stimulated SDH activity and citrate synthase activity by activation of SIRT3, rather than SIRT1 activity, in HepG2 cells (50). Intriguingly, resveratrol has been reported to be an activator of sirtuins, such as SIRT3. Our findings indicated that resveratrol treatment increased SIRT3 expression and decreased GPR91 and α-SMA expression in the liver and isolated HSCs, while improving the NASH phenotype. Honokiol (3',5-di-(2-propenyl)-1,1'-biphenyl-2,4'-diol) is a bioactive natural bi-phenolic compound derived from Magnolia trees. Recently, Gupta and colleagues demonstrated that honokiol bound to SIRT3 physically and increased deacetylase activity of SIRT3 as a SIRT3 activator in myocardium (51). Our study showed that honokiol treatment

enhanced SIRT3 expression and ameliorated the palmitate- or MCD media-induced HSC activation via SIRT3-SDH-GPR91 pathway in LX2 cells.

Several recent studies reported crosstalk between hepatocytes and HSCs as a means of mediating the progression of hepatic fibrosis (52,53). Hui Qian et al. demonstrated that hepatocyte nuclear factor 1α (HNF1-α) suppression in hepatocytes enhanced the activation of HSCs suggesting the presence of an HNF1–α modulated feedback circuit between hepatocytes and HSCs (52). Another study showed that the free fatty acid-induced HSC activation was significantly enhanced in a simultaneous co-culture system with hepatocytes and LX2 cells in the same plate while this effect was absent in both single cells and Transwell model, suggesting the cell-to-cell interaction (53).

Our study showed that the decreased activity of SDH with concurrent increased succinate levels in the CM of palmitate-treated hepatocytes may enhance the activation of HSCs, suggesting that succinate released from hepatocyte modulates HSC activation by a paracrine action. In chronic liver injury, hepatocytes are impaired extensively, which could result in the release of many cytokines, including succinate, and may influence HSC activation through cell-to-cell interactions. Thus, restoration of hepatocyte dysfunction or deactivation of HSC, particularly through SIRT3 activity, may be an effective strategy to prevent NAFLD.

ERK1/2 are members of the mitogen-activated protein kinase (MAPK) family whose activation is linked to cell growth, proliferation, differentiation, motility, and survival (54,55). We found that succinate activated ERK1/2 phosphorylation signaling in LX2 cells. Additionally, pre-treatment with an ERK inhibitor (U0126) significantly blocked the succinate-induced upregulation of GPR91 and α-SMA expression in the LX2 cells suggesting that the ERK pathway is involved in GPR91 signaling. There have been many reports suggesting that restoration of SIRT3 in SIRT3-depleted animals and tissue cultures has a protective effect on mitochondrial metabolism, including fatty acid oxidation and NAFLD development. However, there is presently little direct evidence suggesting that tissue-specific

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SIRT3 overexpression in animal models of NAFLD is protective against insulin resistance. These and other related experiments will be critical for understanding the possible therapeutic value of a SIRT3 agonist. Presently, many questions pivotal to a complete understanding of the potential benefits and pathway of SIRT3-SDH-GPR91 signaling, for the treatment of NAFLD, remain unanswered and require further investigation. However, our study suggested that palmitate treatment of hepatocytes or HSCs decreased SIRT3 activity, thereby decreasing SDH activity and increasing

succinate accumulations due to decreased SDH activity in hepatocytes, or to HSCs moving out from cytosol and activating GPR91 in HSCs causing hepatic fibrogenesis (Fig. 12).

In conclusion, the present investigation showed a novel molecular and cellular mechanism of a SIRT3/SDH/GPR91 cascade in MCD-induced HSC activation in NAFLD. These findings highlight the biological significance of novel strategies targeting SIRT3 and GPR91 in HSCs with the goal of improving NAFLD.

ACKNOWLEDGEMENTS This research was funded by research grant NRF-2013R1A1A1058962. We thank Professor Ki-Up Lee, University of Ulsan College of Medicine for the advice and thank Professor Ja June Jang, Seoul National University for the provision of LX2 cells.

CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.

AUTHOR CONTRIBUTIONS Cho E-H designed the study. Cho E-H and Li

YH co-wrote the paper. Li YH performed most of the

experiments. Seo SR and Lee EH did the immunofluorescent staining regarding the SIRT3 and mitochondrial co-staining. Lee S did the special staining of liver tissue using SIRT3, GPR91 and α-SMA. Cho E-H, Choi DH and Li

YH analyzed all of the experiments. All authors reviewed the results

and approved the final version of the manuscript.

REFERENCES 1. Masuoka, H. C., and Chalasani, N. (2013) Nonalcoholic fatty liver disease: an emerging

threat to obese and diabetic individuals. Annals of the New York Academy of Sciences 1281, 106-122

2. McCullough, A. J. (2006) Pathophysiology of nonalcoholic steatohepatitis. Journal of clinical gastroenterology 40 Suppl 1, S17-29

3. Lim, S. (2014) Ectopic fat assessment focusing on cardiometabolic and renal risk. Endocrinology and metabolism 29, 1-4

4. Yoo, H. J., and Choi, K. M. (2015) Hepatokines as a Link between Obesity and Cardiovascular Diseases. Diabetes & metabolism journal 39, 10-15

5. Targher, G., Day, C. P., and Bonora, E. (2010) Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease. The New England journal of medicine 363, 1341-1350

6. Adams, L. A., Lymp, J. F., St Sauver, J., Sanderson, S. O., Lindor, K. D., Feldstein, A., and Angulo, P. (2005) The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 129, 113-121

7. Ong, J. P., Pitts, A., and Younossi, Z. M. (2008) Increased overall mortality and liver-related mortality in non-alcoholic fatty liver disease. Journal of hepatology 49, 608-612

8. Caldwell, S. H., Oelsner, D. H., Iezzoni, J. C., Hespenheide, E. E., Battle, E. H., and Driscoll, C. J. (1999) Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease. Hepatology (Baltimore, Md.) 29, 664-669

9. Friedman, S. L. (2008) Mechanisms of hepatic fibrogenesis. Gastroenterology 134, 1655-

by guest on Decem

ber 31, 2019http://w

ww

.jbc.org/D

ownloaded from

10

1669 10. Ismail, M. H., and Pinzani, M. (2011) Reversal of hepatic fibrosis: pathophysiological basis

of antifibrotic therapies. Hepatic medicine : evidence and research 3, 69-80 11. Onyango, P., Celic, I., McCaffery, J. M., Boeke, J. D., and Feinberg, A. P. (2002) SIRT3, a

human SIR2 homologue, is an NAD- dependent deacetylase localized to mitochondria. Proceedings of the National Academy of Sciences 99, 13653-13658

12. Cho, E. H. (2014) SIRT3 as a Regulator of Non-alcoholic Fatty Liver Disease. Journal of lifestyle medicine 4, 80-85

13. Schwer, B., North, B. J., Frye, R. A., Ott, M., and Verdin, E. (2002) The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondr ial nicotinamide adenine dinucleotide-dependent deacetylase. The Journal of cell biology 158, 647-657

14. Nogueiras, R., Habegger, K. M., Chaudhary, N., Finan, B., Banks, A. S., Dietrich, M. O., Horvath, T. L., Sinclair, D. A., Pfluger, P. T., and Tschop, M. H. (2012) Sirtuin 1 and sirtuin 3: physiological modulators of metabolism. Physiological reviews 92, 1479-1514

15. Jing, E., Emanuelli, B., Hirschey, M. D., Boucher, J., Lee, K. Y., Lombard, D., Verdin, E. M., and Kahn, C. R. (2011) Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production. Proceedings of the National Academy of Sciences of the United States of America 108, 14608-14613

16. Hirschey, M. D., Shimazu, T., Jing, E., Grueter, C. A., Collins, A. M., Aouizerat, B., Stancakova, A., Goetzman, E., Lam, M. M., Schwer, B., Stevens, R. D., Muehlbauer, M. J., Kakar, S., Bass, N. M., Kuusisto, J., Laakso, M., Alt, F. W., Newgard, C. B., Farese, R. V., Jr., Kahn, C. R., and Verdin, E. (2011) SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome. Molecular cell 44, 177-190

17. Hirschey, M. D., Shimazu, T., Huang, J. Y., Schwer, B., and Verdin, E. (2011) SIRT3 regulates mitochondrial protein acetylation and intermediary metabolism. Cold Spring Harbor symposia on quantitative biology 76, 267-277

18. Hirschey, M. D., Shimazu, T., Goetzman, E., Jing, E., Schwer, B., Lombard, D. B., Grueter, C. A., Harris, C., Biddinger, S., Ilkayeva, O. R., Stevens, R. D., Li, Y., Saha, A. K., Ruderman, N. B., Bain, J. R., Newgard, C. B., Farese, R. V., Alt, F. W., Kahn, C. R. and Verdin, E. (2010) SIRT3 regulates fatty acid oxidation via reversible enzyme deacetylation. Nature 464,121-125

19. Lombard, D. B., Alt, F. W., Cheng, H. L., Bunkenborg, J., Streeper, R. S., Mostoslavsky, R., Kim, J., Yancopoulos, G., Valenzuela, D., Murphy, A., Yang, Y., Chen, Y., Hirschey, M. D., Bronson, R. T., Haigis, M., Guarente, L. P., Farese, R. V., Jr., Weissman, S., Verdin, E., and Schwer, B. (2007) Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation. Molecular and cellular biology 27, 8807-8814

20. Kendrick, A. A., Choudhury, M., Rahman, S. M., McCurdy, C. E., Friederich, M., Van Hove, J. L., Watson, P. A., Birdsey, N., Bao, J., Gius, D., Sack, M. N., Jing, E., Kahn, C. R., Friedman, J. E., and Jonscher, K. R. (2011) Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation. The Biochemical journal 433, 505-514

21. He, J., Hu, B., Shi, X., Weidert, E. R., Lu, P., Xu, M., Huang, M., Kelley, E. E., and Xie, W. (2013) Activation of the aryl hydrocarbon receptor sensitizes mice to nonalcoholic steatohepatitis by deactivating mitochondrial sirtuin deacetylase Sirt3. Molecular and cellular biology 33, 2047-2055

22. Wu, T., Liu, Y. H., Fu, Y. C., Liu, X. M., and Zhou, X. H. (2014) Direct evidence of sirtuin downregulation in the liver of non-alcoholic fatty liver disease patients. Annals of clinical and laboratory science 44, 410-418

23. Cimen, H., Han, M. J., Yang, Y., Tong, Q., Koc, H., and Koc, E. C. (2010) Regulation of succinate dehydrogenase activity by SIRT3 in mammalian mitochondria. Biochemistry 49, 304-311

24. Finley, L. W., Haas, W., Desquiret-Dumas, V., Wallace, D. C., Procaccio, V., Gygi, S. P., and

by guest on Decem

ber 31, 2019http://w

ww

.jbc.org/D

ownloaded from

11

Haigis, M. C. (2011) Succinate dehydrogenase is a direct target of sirtuin 3 deacetylase activity. PloS one 6, e23295

25. Ariza, A. C., Deen, P. M., and Robben, J. H. (2012) The succinate receptor as a novel therapeutic target for oxidative and metabolic stress-related conditions. Frontiers in endocrinology 3, 22

26. Krebs, H. A. (1970) Rate control of the tricarboxylic acid cycle. Advances in enzyme regulation 8, 335-353

27. He, W., Miao, F. J., Lin, D. C., Schwandner, R. T., Wang, Z., Gao, J., Chen, J. L., Tian, H., and Ling, L. (2004) Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature 429, 188-193

28. Regard, J. B., Sato, I. T., and Coughlin, S. R. (2008) Anatomical profiling of G protein-coupled receptor expression. Cell 135, 561-571

29. Robben, J. H., Fenton, R. A., Vargas, S. L., Schweer, H., Peti-Peterdi, J., Deen, P. M., and Milligan, G. (2009) Localization of the succinate receptor in the distal nephron and its signaling in polarized MDCK cells. Kidney international 76, 1258-1267

30. Gnana-Prakasam, J. P., Ananth, S., Prasad, P. D., Zhang, M., Atherton, S. S., Martin, P. M., Smith, S. B., and Ganapathy, V. (2011) Expression and iron-dependent regulation of succinate receptor GPR91 in retinal pigment epithelium. Investigative ophthalmology & visual science 52, 3751-3758

31. Sapieha, P., Sirinyan, M., Hamel, D., Zaniolo, K., Joyal, J. S., Cho, J. H., Honore, J. C., Kermorvant-Duchemin, E., Varma, D. R., Tremblay, S., Leduc, M., Rihakova, L., Hardy, P., Klein, W. H., Mu, X., Mamer, O., Lachapelle, P., Di Polo, A., Beausejour, C., Andelfinger, G., Mitchell, G., Sennlaub, F., and Chemtob, S. (2008) The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis. Nature medicine 14, 1067-1076

32. Li, Y. H., Woo, S. H., Choi, D. H., and Cho, E. H. (2015) Succinate causes alpha-SMA production through GPR91 activation in hepatic stellate cells. Biochemical and biophysical research communications 463, 853-858

33. Friedman, S. L. (1993) Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. The New England journal of medicine 328, 1828-1835

34. Knittel, T., Janneck, T., Muller, L., Fellmer, P., and Ramadori, G. (1996) Transforming growth factor beta 1-regulated gene expression of Ito cells. Hepatology (Baltimore, Md.) 24, 352-360

35. Ahn, B. H., Kim, H. S., Song, S., Lee, I. H., Liu, J., Vassilopoulos, A., Deng, C. X., and Finkel, T. (2008) A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proceedings of the National Academy of Sciences of the United States of America 105, 14447-14452

36. Lantier, L., Williams, A. S., Williams, I. M., Yang, K. K., Bracy, D. P., Goelzer, M., James, F. D., Gius, D., and Wasserman, D. H. (2015) SIRT3 is crucial for maintaining skeletal muscle insulin action and protects against severe insulin resistance in high fat fed mice. Diabetes 64(9):3081-92

37. Bao, J., Scott, I. , Lu, Z., Pang, L., Dimond, C. C., Gius, D., and Sack, M. N. (2010) SIRT3 is regulated by nutrient excess and modulates hepatic susceptibility to lipotoxicity. Free radical biology & medicine 49, 1230-1237

38. Lukyanova LD, Kirova YI. (2015) Mitochondria-controlled signaling mechanisms of brain protection in hypoxia. Front Neurosci. 9:320.

39. Toma, I., Kang, J. J., Sipos, A., Vargas, S., Bansal, E., Hanner, F., Meer, E., and Peti-Peterdi, J. (2008) Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney. The Journal of clinical investigation 118, 2526-2534

40. Vargas, S. L., Toma, I., Kang, J. J., Meer, E. J., and Peti-Peterdi, J. (2009) Activation of the succinate receptor GPR91 in macula densa cells causes renin release. Journal of the American Society of Nephrology : JASN 20, 1002-1011

41. Peti-Peterdi, J. (2010) High glucose and renin release: the role of succinate and GPR91.

by guest on Decem

ber 31, 2019http://w

ww

.jbc.org/D

ownloaded from

12

Kidney international 78, 1214-1217 42. Deen, P. M., and Robben, J. H. (2011) Succinate receptors in the kidney. Journal of the

American Society of Nephrology : JASN 22, 1416-1422 43. Correa, P. R., Kruglov, E. A., Thompson, M., Leite, M. F., Dranoff, J. A., and Nathanson, M.

H. (2007) Succinate is a paracrine signal for liver damage. Journal of hepatology 47, 262-269 44. Howitz, K. T., Bitterman, K. J., Cohen, H. Y., Lamming, D. W., Lavu, S., Wood, J. G.,

Zipkin, R. E., Chung, P., Kisielewski, A., Zhang, L. L., Scherer, B., and Sinclair, D. A. (2003) Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425, 191-196

45. Kulkarni, S. S., and Canto, C. (2015) The molecular targets of resveratrol. Biochimica et biophysica acta 1852, 1114-1123

46. Pacholec, M., Bleasdale, J. E., Chrunyk, B., Cunningham, D., Flynn, D., Garofalo, R. S., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. The Journal of biological chemistry 285, 8340-8351

47. Beher, D., Wu, J., Cumine, S., Kim, K. W., Lu, S. C., Atangan, L., and Wang, M. (2009) Resveratrol is not a direct activator of SIRT1 enzyme activity. Chemical biology & drug design 74, 619-624

48. Pan, Q. R., Ren, Y. L., Liu, W. X., Hu, Y. J., Zheng, J. S., Xu, Y., and Wang, G. (2015) Resveratrol prevents hepatic steatosis and endoplasmic reticulum stress and regulates the expression of genes involved in lipid metabolism, insulin resistance, and inflammation in rats. Nutrition research (New York, N.Y.) 35, 576-584

49. Timmers, S., Konings, E., Bilet, L., Houtkooper, R. H., van de Weijer, T., Goossens, G. H., Hoeks, J., van der Krieken, S., Ryu, D., Kersten, S., Moonen-Kornips, E., Hesselink, M. K., Kunz, I., Schrauwen-Hinderling, V. B., Blaak, E. E., Auwerx, J., and Schrauwen, P. (2011) Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans. Cell metabolism 14, 612-622

50. Desquiret-Dumas, V., Gueguen, N., Leman, G., Baron, S., Nivet-Antoine, V., Chupin, S., Chevrollier, A., Vessieres, E., Ayer, A., Ferre, M., Bonneau, D., Henrion, D., Reynier, P., and Procaccio, V. (2013) Resveratrol induces a mitochondrial complex I-dependent increase in NADH oxidation responsible for sirtuin activation in liver cells. The Journal of biological chemistry 288, 36662-36675

51. Pillai VB, Samant S, Sundaresan NR, Raghuraman H, Kim G, Bonner MY, Arbiser JL, Walker DI, Jones DP, Gius D, Gupta MP. (2015) Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3. Nat Commun. 14;6:6656

52. Qian, H., Deng, X., Huang, Z. W., Wei, J., Ding, C. H., Feng, R. X., Zeng, X., Chen, Y. X., Ding, J., Qiu, L., Hu, Z. L., Zhang, X., Wang, H. Y., Zhang, J. P., and Xie, W. F. (2015) An HNF1alpha-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells. Cell research 25, 930-945

53. Barbero-Becerra, V. J., Giraudi, P. J., Chavez-Tapia, N. C., Uribe, M., Tiribelli, C., and Rosso, N. (2015) The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH. Toxicology in vitro : an international journal published in association with BIBRA 29, 1753-1758

54. Cobb, M. H., Hepler, J. E., Cheng, M., and Robbins, D. (1994) The mitogen-activated protein kinases, ERK1 and ERK2. Seminars in cancer biology 5, 261-268

55. Roux, P. P., and Blenis, J. (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiology and molecular biology reviews : MMBR 68, 320-344

FIGURE LEGENDS Figure 1. Succinate, succinate dehydrogenase activity and expression of G protein-coupled

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receptor 91 (GPR91) and alpha-smooth muscle actin (α-SMA) in LX2 cells. (A) LX2 cells were cultured in the presence or absence of succinate (400 uM) for 24 h and U1026 was added 2h before harvest. Whole-cell lysates were subjected to western blot analyses with the indicated antibodies (left). Band intensities were calculated using the Image J software (National Institutes of Health [NIH], Bethesda, MD, USA) (right). ***P < 0.001, vs. control. (B) LX2 cells were cultured in the presence or absence of malonate (3 mM) for 24 h and GPR91 and a-SMA protein levels were analyzed using western blotting (top). Band intensities were calculated using Image J software (NIH) (bottom). ***P < 0.001, vs. control. (C) LX2 cells were cultured in control or malonate (3 mM) for 24 h and SDH activity was measured in whole-cell lysates. ***P < 0.001, vs. control. (D) LX2 cells were cultured in the presence or absence of malonate (3 mM) for 24 h and succinate concentrations were measured in whole-cell lysates. ***P < 0.001, vs. control. (E) LX2 cells were cultured in the presence or absence of D-malate (1 mM) for 24 h and GPR91 and a-SMA protein levels were analyzed using western blotting (top). Band intensities were calculated using Image J software (NIH) (bottom). ***P < 0.001, vs. control. (F) LX2 cells were cultured in the presence or absence of D-malate (1 mM) for 24 h and fumarase activity was measured in whole-cell lysates. ***P < 0.001, vs. control. (G) LX2 cells were cultured in the presence or absence of D-malate (1 mM) for 24 h and SDH activity was measured in whole-cell lysates. ***P < 0.001, vs. control. (H) LX2 cells were cultured in the presence or absence of D-malate (1 mM) for 24 h and succinate concentrations were measured in whole-cell lysates. ***P < 0.001, vs. control. Figure 2. Upregulation of GPR91, α-SMA, and TGF β1, and collagen type 1 in LX2 cells by

siRNA-mediated knockdown of Sirtuin 3 (SIRT3). (A) RT-PCR analysis of SIRT3, GPR91, α-SMA, TGF β1, collagen type I mRNA expression in hepatic stellate cells (HSCs) transfected with SIRT3 siRNA or control siRNA. (B) Quantitative analysis of the band density. Each column represents means ± SEM. **P < 0.01, ***P < 0.001, vs. control. Figure 3. SIRT3 regulation of succinate dehydrogenase (SDH)-GPR91-α-SMA signaling

pathway. (A) Western blot analysis of SIRT3, GPR91, α-SMA, TGF β1, and collagen type I protein expression in LX2 cells with or without SIRT3 knockdown. The level was normalized to the expression of GAPDH (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ). ***P < 0.001, vs. control siRNA. (B) LX2 cells were transfected with SIRT3 siRNA or Control siRNA for 24 h and SDH activity was measured in whole cell lysates. ***P< 0.001, vs. control siRNA. (C) LX2 cells were transfected with SIRT3 siRNA or control siRNA for 24 h and succinate concentration was measured in whole-cell lysates. ***P < 0.001, vs. control siRNA. (D) LX2 cells were transfected with SIRT3 siRNA or control siRNA for 24 h. Then, the LX2 cells were immunoprecipitated with monoclonal antibodies against SDHA. SDHA acetylation was examined using immunoblotting (left). Band intensities were calculated using the ImageJ software (NIH) (right). ***P < 0.001, vs. control siRNA (E) Overexpression of SIRT3 by transfection with human SIRT3 adenovirus (Ad-SIRT3) or control adenovirus expressing LacZ (Ad-CMV-𝛽-gal) at a MOI of 30. After 24 h, infected LX2 cells were lysed and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control adenovirus. (F) LX2 cells were transfected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. After 24 h, infected cells were lysed and SDH activity was measured. ***P < 0.001, vs. control adenovirus. (G) LX2 cells were transfected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. After 24 h, infected cells were lysed and succinate concentrations were measured. ***P < 0.001, vs. control adenovirus. (H) LX2 cells were transfected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. After 24 h,

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infected cells were immunoprecipitated with monoclonal antibodies against SDHA. SDHA acetylation was examined using immunoblotting (left). Band intensities were calculated using the ImageJ software (NIH) (right). ***P < 0.001, vs. control adenovirus. Figure 4. Effects of palmitate- and methionine- and choline- deficient (MCD) media on SIRT3

and GPR91 expression in LX2 cells. (A) LX2 cells were treated with or without palmitate for 20 h. Subsequently, SIRT3, GPR91 and α-SMA were detected using western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control. (B) LX2 cells were treated with or without palmitate for 20 h and SIRT3 mRNA was detected using RT-PCR analysis (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). . ***P < 0.001, vs. control. (C) LX2 cells were cultured in control or MCD media for 24 h. Cell lysates were prepared and SIRT3, GPR91, and α-SMA protein levels were analyzed using western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). *P < 0.05, ***P < 0.001, vs. control media. (D) LX2 cells were cultured in control or MCD media for 24 h and SIRT3 mRNA was detected using RT-PCR analysis (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control media. Figure 5. SIRT3 overexpression attenuates palmitate-and MCD media-induced HSC activation.

(A) LX2 cells were transfected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. After 8 h, infected cells were incubated with or without palmitate (300 μM) for 20 h, and then cells were lysed and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). *P < 0.05, **P < 0.01, ***P < 0.001, vs. control. (B) LX2 cells were transfected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. After 8 h, infected cells were incubated with or without palmitate (300 μM) for 20 h, and SDH activity was measured in whole cell lysates. **P < 0.01, ***P < 0.001, vs. control. (C) LX2 cells were transfected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. After 8 h, infected cells were incubated with or without palmitate (300 μM) for 20 h, and succinate concentrations were measured in whole-cell lysates. ***P < 0.001, vs. control. (D) Before transfection, LX2 cells were changed to control or MCD media. Then LX2 cells were

infected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. At 24 h post-infection, cells were lysed and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). *P < 0.05, **P < 0.01, ***P < 0.001, vs. control. (E) Before transfection, LX2 cells were changed to control or MCD media. Then LX2 cells were

infected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. At 24 h post-infection, SDH activity was measured in whole-cell lysates. **P < 0.01, vs. control media. (F) Before transfection, LX2 cells were changed to control or MCD media. Then LX2 cells were

infected with Ad-SIRT3 or Ad-CMV-𝛽-gal at a MOI of 30. At 24 h post-infection, succinate concentrations were measured in whole-cell lysates. ***P < 0.001, vs. control media. Figure 6. Honokiol attenuates palmitate- and MCD media-induced HSC activation through

SIRT3-SDH-Succinate pathway. (A) LX2 cells were treated with or without honokiol (10 μM). After 4 h, the cells were incubated with or without palmitate (300 μM) for 20 h, and then cells were lysed and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control. (B) LX2 cells were treated with or without honokiol (10 μM). After 4 h, the cells were incubated with or without palmitate (300 μM) for 20 h, and SDH activity was measured in whole cell lysates. ***P < 0.001, vs. control. (C) LX2 cells were treated with or without honokiol(10 μM). After 4 h, the cells were incubated with or without palmitate (300 μM) for 20 h, and succinate concentrations were measured in whole-cell lysates. ***P < 0.001, vs. control.

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(D) LX2 cells were changed to control or MCD media. Then LX2 cells were treated with or without honokiol (10 μM) for 24 h, and cells were lysed and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control. (E) LX2 cells were changed to control or MCD media. Then LX2 cells were treated with or without honokiol (10 μM) for 24 h, and SDH activity was measured in whole-cell lysates. ***P < 0.001, vs. control media. (F) LX2 cells were changed to control or MCD media. Then LX2 cells were treated with or without honokiol (10 μM) for 24 h, and succinate concentrations were measured in whole-cell lysates. ***P < 0.001, vs. control media. Figure 7. Adeno-associated virus (AAV)-GPR91 shRNA-mediated knockdown of GPR91 alters

α-SMA expression in isolated HSCs and liver from MCD diet-fed mice model of NAFLD. (A) MCD diet-fed mice treated with AAV-GPR91 shRNA or AAV6-GFP (control shRNA) at the first day of MCD diet feeding and hepatic steatosis evaluated with H&E staining. (B) MCD diet-fed mice treated with AAV-GPR91 shRNA or AAV6-GFP (control shRNA) at the first day of MCD diet feeding; Masson’s trichrome staining was also performed. (C) MCD diet-fed mice treated with AAV-GPR91 shRNA or AAV6-GFP (control shRNA) at the first day of MCD diet feeding; the expression of GPR91 was evaluated by immunohistochemistry. (D) MCD diet-fed mice treated with AAV-GPR91 shRNA or AAV6-GFP (control shRNA) at the first day of MCD diet feeding; the expression of α-SMA was evaluated by immunohistochemistry. (E) MCD diet-fed mice treated with AAV-GPR91 shRNA or AAV6-GFP (control shRNA) at the first day of MCD diet feeding; the liver were lysed and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. chow diet. (F) MCD diet-fed mice treated with AAV-GPR91 shRNA or AAV6-GFP (control shRNA) at the first day of MCD diet feeding; HSC cells were isolated and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). **P < 0.01, ***P < 0.001, vs. chow diet. Figure 8. Effect of resveratrol on GPR91 and α-SMA expression in isolated HSCs and liver from

MCD diet-fed mice model of NAFLD. (A) MCD diet-fed mice treated with or without resveratrol; hepatic steatosis was evaluated with H&E staining. (B) MCD diet-fed mice treated with or without resveratrol; Masson’s trichrome staining was performed. (C) MCD diet-fed mice treated with or without resveratrol; the expression of GPR91 was evaluated by immunohistochemistry. (D) MCD diet-fed mice treated with or without resveratrol; the expression of α-SMA was evaluated by immunohistochemistry. (E) MCD diet-fed mice treated with or without resveratrol; the liver was lysed and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). *P < 0.05, **P < 0.01, ***P < 0.001, vs. chow diet. (F) MCD diet-fed mice treated with or without resveratrol; HSC cells were isolated and subjected to western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). *P < 0.05, ***P < 0.001, vs. chow diet. Figure 9. Effect of resveratrol on SIRT3 expression in liver from MCD diet-fed mice model of

NAFLD. (A) MCD diet-fed mice treated with or without resveratrol; the expression of SIRT3 was evaluated by immunohistochemistry. (B) Representative immunofluorescent double staining for HSP60 (green) and SIRT3 (red). Merged images are at the right. (C) MCD diet-fed mice treated with AAV-GPR91 shRNA or resveratrol and triglyceride (TG) contents in the liver were determined. **P < 0.01, ***P < 0.001, vs. chow diet.

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Figure 10. Effect of palmitate and MCD media treatment on hepatocytes isolated from chow

diet-fed mice. (A) Primary hepatocytes isolated from the livers of chow diet-fed mice were treated with or without palmitate (300 μM) for 20 h and SIRT3 protein levels were determined using western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control. (B) Primary hepatocytes isolated from the livers of chow diet-fed mice were treated with or without palmitate (300 μM) for 20 h and SDH activity was measured in whole-cell lysates. **P < 0.01, vs. control. (C) Primary hepatocytes isolated from the livers of chow diet-fed mice were treated with or without palmitate (300 μM) for 20 h and succinate concentrations were measured in whole-cell lysates. ***P < 0.001, vs. control. (D) Primary hepatocytes isolated from the livers of chow diet-fed mice were cultured in control or MCD media for 24 h and SIRT3 protein levels were determined using western blotting (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control media. (E) Primary hepatocytes isolated from the livers of chow diet-fed mice were cultured in control or MCD media for 24 h and SDH activity was measured in whole cell lysates. ***P < 0.001, vs. control media. (F) Primary hepatocytes isolated from the livers of chow diet-fed mice cultured in control or MCD media for 24 h and succinate concentration was measured in whole cell lysates. ***P < 0.001, vs. control media.

Figure 11. Effect of conditioned media (CM) from hepatocytes exposed to palmitate on HSC

activation in vitro.

(A) Schematic representation of cell to cell experiments with mouse hepatocytes and hepatic stellate cells. (B) AML12 cells were treated with or without palmitate (300 μM) for 20 h and SDH activity was measured. ***P < 0.001, vs. control. (C) AML12 cells were treated with or without palmitate (300 μM) for 20 h and succinate concentrations were measured. ***P< 0.001, vs. control. (D) Conditioned media (CM) from palmitate-treated AML12 cells was transferred to LX2 cells for 20 h and western blot analysis was performed with indicated antibodies in LX2 cells (top). Band intensities were calculated using the ImageJ software (NIH) (bottom). ***P < 0.001, vs. control. (E) Conditioned media (CM) from palmitate-treated AML12 cells was transferred to LX2 cells for 20 h and SDH activity was measured in whole-cell lysates of LX2 cells. ***P < 0.001, vs. control. (F) Conditioned media (CM) from palmitate-treated AML12 cells was transferred to LX2 cells for 20 h and succinate concentrations were measured in whole cell lysates of LX2 cells. ***P < 0.001, vs. control. Figure 12. SIRT3, SDH, and GPR91 signaling pathway in HSCs and hepatocytes. Each solid line and arrow denotes a step in an activating pathway and the dashed line and arrow denote a step in an inhibiting pathway. Palmitate decreases SIR3 activity, thereby decreasing SDH activity in hepatocytes and HSCs. Increased succinate accumulation from decreased SDH activity in hepatocytes or HSCs moves out from the cytosol and activates GPR91 in HSCs, resulting to the

production of α-SMA, TGF β1, and collagen type I proteins.

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Figure 2. Upregulation of GPR91,α-SMA and TGF β1, and collagen

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Figure 3. SIRT3 Regulation of SDH-GPR91- α-SMA

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Figure 7. AAV-GPR91shRNA-Mediated Knockdown of GPR91 Alters

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Figure 8. Effect of Resveratrol on GPR91and α-SMA Expression in

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Resvera

tro

l

100x 400x

α-SMA staining

A

MC

D d

iet+

Resvera

tro

l M

CD

die

t C

ho

w d

iet

40x 200x

H&E staining

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Figure 9. Effect of Resveratrol on SIRT3 Expression in Liver from

MCD Diet Fed Mice Model of NAFLD

0

20

40

60

80

100

120Chow dietMCD dietMCD diet+ ResveratrolMCD diet+ AAV6-GFPshRNAMCD diet+ AAV-GPR91shRNA

Hep

ati

c T

G (

mg

/g)

***

**

***

***

C

Ch

ow

die

t M

CD

die

t M

CD

die

t+

Resvera

tro

l

SIRT3 staining

400x 100x

A B

Ch

ow

die

t M

CD

die

t M

CD

die

t+

Resvera

tro

l

HSP 60 SIRT3 Merge

IF co- staining 1000x

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F

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

***

Su

ccin

ate

co

nc.(

fold

)

Hepatocyte

Control media

MCD media

Hepatocyte 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

E

***

Rela

tiv

e a

cti

vit

y o

f S

DH

Control media

MCD media

D

SIRT3

GAPDH

MC

D m

ed

ia

Co

ntr

ol m

ed

ia

A P

alm

itate

GAPDH

SIRT3

Co

ntr

ol

0

0.5

1

1.5

2Control media

MCD media

***

Pro

tein

levels

(fo

ld)

Hepatocyte 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6ControlPalmitate

C

***

Su

ccin

ate

co

nc.(

fold

)

***

0

0.5

1

1.5

2

2.5Control

Palmitate

Pro

tein

levels

(fo

ld)

Figure 10. Effect of Palmitate and MCD Media Treatment

on Hepatocytes Isolated from Chow Diet Fed Mice

** R

ela

tiv

e a

cti

vit

y o

f S

DH

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6Control

B

Hepatocyte

Palmitate

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GPR91

Figure 11. Effect of Conditioned Media from Hepatocytes

Exposed to Palmitate on HSC Activation in vitro

AML12 cells

(hepatocytes)

Palmitate

treatment (20 h)

Conditional media (CM)

from AML12 cells

SDH activity

measurement (B)

Succinate

measurement (C)

CM Treatment on

LX2 cells for 20 h

Western blotting of

SIRT3, GPR91, α-SMA(D)

Measurement of SDH

activity (E), Succinate (F)

A

Conditional

media(CM)

+ -

α-SMA

SIRT3

D

LX2

***

Pro

tein

levels

(fo

ld)

0

0.5

1

1.5

2

2.5Control media

CM media

***

***

***

GAPDH

***

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6 Control

Palmitate

B

Rela

tiv

e a

cti

vit

y o

f S

DH

AML12 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2 Control

Palmitate

C

AML12 S

uccin

ate

co

nc.(

fold

)

***

0

0.5

1

1.5

2

2.5 Control

Palmitate

F

LX2

Su

ccin

ate

co

nc.(

fold

) ***

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

E

Rela

tiv

e a

cti

vit

y o

f S

DH

***

LX2

Palmitate

Control

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Figure 12. SIRT3, SDH and GPR91 Signaling Pathway in the

Hepatic stellate cells and Hepatocyte

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ChoYing Hui Li, Dae Hee Choi, Eun Hye Lee, Su Ryeon Seo, Seungkoo Lee and Eun-Hee

pathway in hepatic stellate cells-SMA production through the succinate dehydrogenase-GPR91αSIRT3 regulates

published online February 24, 2016J. Biol. Chem. 

  10.1074/jbc.M115.692244Access the most updated version of this article at doi:

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